CN110331221A - Plasmodium gene diagnostic primers - Google Patents

Plasmodium gene diagnostic primers Download PDF

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CN110331221A
CN110331221A CN201910611312.9A CN201910611312A CN110331221A CN 110331221 A CN110331221 A CN 110331221A CN 201910611312 A CN201910611312 A CN 201910611312A CN 110331221 A CN110331221 A CN 110331221A
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杨照青
陈熙
张家祺
徐士玲
耿劲婷
黄亚铭
黄超
曾炜林
向征
杨照坤
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Kunming Medical University
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Abstract

The present invention discloses a kind of plasmodium gene diagnostic primers, the primer is ring mediated isothermal amplification (LAMP) primer that whether there is specific Plasmodium and three kinds of plasmodiums in detection/identification sample, according to the present invention, use the primer sets, it simultaneously or separately can detect or identify the infection of one of Plasmodium and/or plasmodium falciparum, Plasmodium vivax, Plasmodium ovale or a variety of plasmodiums, the quality time can be won for the diagnosis and treatment of Chinese malaria, to realize that prevention and control and the elimination of malaria prevalence provide help.

Description

Plasmodium gene diagnostic primers
Technical field
The present invention relates to biological detections and identification field more particularly to one kind can quickly and correctly detect and identification malaria The primer sets of protozoan infection include Plasmodium, malignant malaria, the primer sets of tertian fever and ovale malaria Infect And Diagnose.
Background technique
In the country of malaria prevalence, quickly and correctly Malaria Diagnosis is an arduous challenge.Different plasmodiums are drawn The malaria clinical manifestation risen is different, and therapeutic scheme is different, and therefore, malaria diagnosis requires to be accurate to worm kind.
In China, many input-response relations infection are related to two or more worm kinds, these mixed infections often ignore or by Underestimate.The failure of mixed infection detection causes malaria treatment insufficient, and may cause the exacerbation and serious complication of disease. Therefore, it needs to develop a kind of feasible, easy, quick, highly sensitive and strong species specificity malaria diagnosis method.
Currently, the diagnostic method of clinically used malaria is the microscope inspection of blood film.If parasite density is very high, this Kind Microscopical Method For Detection has relatively high sensitivity and specificity and can determine the stage of development of plasmodium kind and polypide.But it should Comparison between detecting methods are cumbersome, heavy workload, and trained professional is needed to operate, especially low parasitemia densities, by controlling Malaria diagnosis after treatment is very high to skills involved in the labour requirement, and the probability for mistaken diagnosis occur is big, especially logical in plasmodium density Normal lower Endemic Area, mistaken diagnosis frequently result in the delay in treatment.The area that can not carry out is diagnosed in some standard tests, in order to The speed and accuracy of malaria diagnosis are improved, researchers develop the malaria rapid diagnostic assay (RDT) based on immune response. However, the sensitivity between product has fluctuation, and the product only to the species-specific diagnosis of plasmodium falciparum.Therefore, this Situation can lead to the result or insecure kind of diagnosis of false negative.
Later, researcher developed the molecular biology method based on DNA cloning technology for malaria diagnosis, such as nest Formula PCR and real-time quantitative PCR.Compared with microscope, these methods be proved to sensitivity with higher for mixed infection and Preferable specificity.However, time-consuming for this diagnostic method, and it is expensive, need perfect experiment detection device and profession Technical staff, to limit application of this technology in malaria endemic area, Basic medical and health institutions.
Ring mediated isothermal amplification method (Loop-mediated isothermal amplification, LAMP) is in recent years A kind of constant temperature nucleic acid amplification technology that new development is got up.LAMP method is characterized in for six sections design on target dna chain Then four different primers recycle strand replacement reaction to be reacted at a certain temperature.Reaction only need gene template, Primer, strand displacement type DNA synzyme, matrix etc. are collectively disposed under certain temperature (60~65 DEG C), can be completed through a step. Amplification efficiency is high, can realize 10 in 15 minutes to 1 hour9-1010Amplification again, achievees the effect that monitor.Therefore, Expensive PCR instrument is not needed, only needs simple constant temperature facility such as water-bath that can complete.As a result interpretation can visually be seen Examine turbidity fluorescent dye be perhaps added in the product observed by color change as a result, simple ultraviolet lamp or from It is observed under right light, it is not necessary to additionally increase step.It has simple, quick, High sensitivity, high specificity, without special Equipment, the feature of less demanding to template.These unique advantages receive it can more by numerous medical personnels, and be pushed away Extensively.Suitable with PCR, the parasitemia densities of detection are lower.Requirement of this method for operator is low, can be complete without special training At.
It is higher without sensibility, can be easily in development at the basic level application currently, domestic market is in addition to amynologic diagnostic method Clinical quick diagnosis reagent kit.
Summary of the invention
To solve the above problems, needing to develop the plasmodium sense for meeting China's epidemic characteristic sensitive, special, easy to operate Diagnostic method is contaminated, this method is different from other common malaria diagnosis methods such as Microscopical Method For Detection, immunology diagnosis method and PCR method.
The object of the present invention is to provide sensitive, efficient, easy to operate LAMP diagnostic primers, for determining in people's blood specimen Whether have Infected With Plasmodium or determine whether for malignant malaria, tertian fever or ovale malaria three specific worm kinds it is any one or more of Infection can win the quality time for the diagnosis and treatment of Chinese malaria endemic area, to realize that prevention and control and the elimination of malaria provide It helps.
In order to solve the above problem, present invention application constant temperature nucleic acid amplification technology --- ring mediated isothermal amplification (LAMP) technology Realize the purpose of hypersensitivity and specific detection Infected With Plasmodium.By searching for Plasmodium and plasmodium falciparum, every other day Plasmodium, Plasmodium ovale high special, conservative gene nucleotide series, using relevant primer design software design primer, And by manual analysis, experimental verification, the best primer sets for providing high degree of specificity and sensitivity are finally screened.Application invention The plasmodium gene diagnostic primers group of people's design, can quickly, it is simple, accurately detected whether Plasmodium species parasites sense Dye, and be capable of determining whether as a certain or a variety of parasitic infection in plasmodium falciparum, Plasmodium vivax, Plasmodium ovale.
That is, the present invention can provide the contents of the 1st to 6 description of subordinate.
1. plasmodium gene diagnostic primers for simultaneously or separately detect or identify Plasmodium and/or plasmodium falciparum, The infection of one of Plasmodium vivax, Plasmodium ovale or a variety of plasmodiums；Plasmodium gene diagnostic primers are containing SEQ 1 The oligonucleotides group of the nucleic acid sequence indicated to 6, for expanding the specific region of Plasmodium mtDNA gene order；Contain SEQ The oligonucleotides group of 7 to 12 nucleic acid sequences indicated, the specific region for expanding plasmodium protozoon ActinI gene order；Contain The oligonucleotides group for the nucleic acid sequence for thering is SEQ 13 to 18 to indicate, for expanding the spy of Plasmodium vivax 18S rRNA gene order Determine region；Oligonucleotides group containing the nucleic acid sequence indicated of SEQ19 to 24, for expanding Plasmodium ovale SSU rRNA gene The specific region of sequence.
2. the purposes according to the 1st, detects using a primer sets or identifies the infection of Plasmodium, the primer Group includes the oligonucleotides group containing the nucleic acid sequence indicated of SEQ 1 to 6, for expanding Plasmodium mtDNA gene order Specific region.
3. the purposes according to the 1st, is detected using a primer sets or identifies the infection of plasmodium falciparum, this draws Object group includes the oligonucleotides group containing the nucleic acid sequence indicated of SEQ 7 to 12, is used for expanding plasmodium protozoon ActinI gene The specific region of sequence.
4. the purposes according to the 1st, is detected using a primer sets or identifies the infection of Plasmodium vivax, this draws Object group includes the oligonucleotides group containing the nucleic acid sequence indicated of SEQ 13 to 18, for expanding Plasmodium vivax 18S rRNA base Because of the specific region of sequence.
5. the purposes according to the 1st, detects using a primer or identifies the infection of Plasmodium ovale, the primer Group includes the oligonucleotides group containing the nucleic acid sequence indicated of SEQ 19 to 24, for expanding Plasmodium ovale SSU rRNA gene The specific region of sequence.
6, plasmodium gene diagnostic primers, the oligonucleotides group containing the nucleic acid sequence indicated of SEQ 1 to 6；Contain SEQ 7 The oligonucleotides group of the nucleic acid sequence indicated to 12；Oligonucleotides group containing the nucleic acid sequence indicated of SEQ 13 to 18；Contain The oligonucleotides group for the nucleic acid sequence that SEQ 19 to 24 is indicated.
Beneficial effects of the present invention
The present invention allows using LAMP primer group, and wherein the primer sets include to contain the nucleic acid sequence indicated of SEQ 1 to 6 Oligonucleotides group, for expanding the specific region of Plasmodium mtDNA gene order；The nucleic acid sequence indicated containing SEQ 7 to 12 The oligonucleotides group of column, the specific region for expanding plasmodium protozoon ActinI gene order；It is indicated containing SEQ 13 to 18 Nucleic acid sequence oligonucleotides group, for expanding the specific region of Plasmodium vivax 18S rRNA gene order；Contain SEQ The oligonucleotides group of 19 to 24 nucleic acid sequences indicated, for expanding the given zone of Plasmodium ovale SSU rRNA gene order Domain.To allow simultaneously or separately to detect or distinguish the presence or absence of one of anyone Infected With Plasmodium or three kinds of plasmodiums.
Using Plasmodium of the invention or detection/identification method of three kinds of plasmodiums, it is able to detect malaria infection patient Use the therapeutic effect of malaria treatment drug.
Detailed description of the invention
Fig. 1 shows the position of LAMP target, Plasmodium (A:mtDNA), plasmodium falciparum (B:Pf ActinI), Day plasmodium (C:Pv18SrRNA), the initiation site of Plasmodium ovale (D:Po SSU rRNA) and Plasmodium mtDNA base Cause, plasmodium falciparum actin gene, Plasmodium vivax 18SrRNA gene, Plasmodium ovale SSU rRNA gene nucleotides sequence Column, the position that primer sets cause site are indicated by an arrow.Wherein, gene nucleotide reference sequences are as follows: Plasmodium mtDNA Gene includes plasmodium falciparum mtDNA gene (GenBank registration number M99416.1), Plasmodium vivax mtDNA gene (GenBank registration number KF68441.1), Plasmodium ovale mtDNA gene (GenBank registration number HQ712052.1, HQ712053.1), malariae mtDNA gene (GenBank registration number AB489194.1), Plasmodium knowlesi mtDNA gene (GenBank registration number AY722797.1), plasmodium falciparum ActinI gene (GenBank registration number XM_001350811.1), Plasmodium vivax 18SrRNA gene (GenBank registration number DQ162604.1), Plasmodium ovale SSU rRNA gene (GenBank Registration number JF894405.1, JF894406.1).
Specific embodiment
The present invention will be described in further detail below with reference to the embodiments of the drawings.
Embodiment 1
It is compared by transferring related special, conservative gene sequence in GeneBank, chooses 200bp to 500bp The conservative gene segment of length goes out multiple groups primer with LAMP Designer 1.15 (PREMIER Biosoft) software design, By manually comparing, testing sieve select highest effect, sensitive primer sets.Sequence specific primers group includes containing 1 to 6 table of SEQ The oligonucleotides group for the nucleic acid sequence shown, for expanding the specific region of Plasmodium mtDNA gene order；Containing SEQ 7 to The oligonucleotides group of 12 nucleic acid sequences indicated, the specific region for expanding plasmodium protozoon ActinI gene order；Contain The oligonucleotides group for the nucleic acid sequence that SEQ 13 to 18 is indicated, for expanding the specific of Plasmodium vivax 18S rRNA gene order Region；Oligonucleotides group containing the nucleic acid sequence indicated of SEQ 19 to 24, for expanding Plasmodium ovale SSU rRNA gene The specific region of sequence, is shown in Table 1 in detail.
1 sequence table of table
Serial number Title Primer sequence (5'to3') Base number
SEQ 1 Mt-F3 TGTCAACTACCATGTTACGAC 21
SEQ 2 Mt-B3 AACGGTCCTAAGGTAGCAA 19
SEQ 3 Mt-FIP TACGGCCCGACGGTAAGATCGTAACCATGCCAACAC 36
SEQ 4 Mt-BIP AGGAGTCTCACACTAGCGACAAAATTCCTTGTCGGGTAATCTC 43
SEQ 5 Mt-LpF CTGAGCACCTTAACTTCCCTAA 22
SEQ 6 Mt-LpB TACACCGTTCATGCAGGAC 19
SEQ 7 Pfact1-F3 GGAGAAGAAGATGTTCAAGC 20
SEQ 8 Pfact1-B3 CCCAATTCGTAACAATACCATG 22
SEQ 9 Pfact1-FIP AACGGAACGAGGTGCATCATTTGTTGACAACGGATCAGG 39
SEQ 10 Pfact1-BIP AGTAGGAAGACCAAAGAATCCAGGATGTGCTTCATCACCAACAA 44
SEQ 11 Pfact1-LpF CTCCTGCAACTCCTGCTT 18
SEQ 12 Pfact1-LpB GTTGGTATGGAAGAGAAAGATGC 23
SEQ 13 PV18S-F3 CTAATTAGCGGTAAGTACGACA 22
SEQ 14 PV18S-B3 AGCCTAGTTCATCTAAGGACA 21
SEQ 15 PV18S-FIP ACCAAACGCATCAGCTATTCGTATGTCGGATTGGATCTGGA 41
SEQ 16 PV18S-BIP TTACTTGGCTTATCGTACCGTTCAGACCTGTTGTTGCCTT 40
SEQ 17 PV18S-LpF CACCGACACGAAGTATAATTGC 22
SEQ 18 PV18S-LpB GCTTCTTAGAGGAACGATGTGT 22
SEQ 19 P0ssu-F3 CGAGTTTCTGACCTATCAGC 20
SEQ 20 P0ssu-B3 GCTGGCACCAGACTTG 16
SEQ 21 P0ssu-FIP GATGTGGTAGCTATTTCTCAGGCTCCCTAACATGGCTATGACGG 44
SEQ 22 P0ssu-BIP GCAGCAGGCGCGTAAATTACAACCATGAAATGGCCTTGT 39
SEQ 23 P0ssu-LpF TCTCCGGAATCGAACTCTAATTC 23
SEQ 24 P0ssu-LpB TCTAAAGAAGAGAGGTAGTGACAAG 25
Embodiment 2
The foundation of LAMP reaction system
1, material and method:
Between 2016 to 2018, to 466 malaria suspected case blood samples.Type strain 3D7, DD2, Plasmodium knowlesi H plants of DNA of Strain (are provided) by MR4/ATCC (Manassas, VA).Toxoplasma, Schistosoma japonicum DNA are by Kunming Medical University Helminth teaching and research room provides.
2, prepared by DNA profiling
Using Roche High Pure PCR Template Preparation Kit, insect-taking blood carries out DNA profiling system It is standby.
Nest-type PRC reaction: primer and reaction condition with reference to delivered document (Sun Lingcong, Zhang Huaxun, Pei Sujian, Xia Jing, Wu Dongni, Lin Wen: it examines the etiological diagnosis analysis world of Hubei Province's the first Introduced cases Plasmodium ovale wallikeri subspecies Medical journal 2016,37:1956-1958.)
3, LAMP reacts
It the use of primer sets include the primer sets containing SEQ 1 to 6；The primer sets of SEQ 7 to 12；The primer of SEQ 13 to 18 Group；The primer sets of SEQ 19 to 24, using LAMP shown in reaction system such as table 2.The position of each primer and nucleotide sequence are aobvious Show in Fig. 1.
Table 2. uses LAMP reaction system
4, amplified reaction
Prepared 12.5 μ L reaction system is placed in PCR instrument or metal bath, the constant temperature 45min at 63 DEG C, then 5min makes enzyme-deactivating to terminate reaction at 80 DEG C.
5, it detects
After amplified reaction, reaction tube is placed in ultraviolet lamp and is observed.If being issued as positive control Green fluorescence is then judged as positive (+), if not issuing fluorescence as negative control, is judged as negative (-).
Embodiment 3
Specific test
Other kind of helminth and different plasmodium kinds are tested, its specificity is analyzed.Use the primer of SEQ 1 to 6 Group；The primer sets of SEQ 7 to 12；The primer sets of SEQ 13 to 18；The primer sets of SEQ 19 to 24 are respectively to Schistosoma japonicum, bow Shape worm DNA carries out LAMP reaction, and result is feminine gender.Use the primer sets of SEQ1 to 6；The primer sets of SEQ 7 to 12；SEQ 13 to 18 primer sets；The primer sets of SEQ 19 to 24 are respectively to five kinds of plasmodium plasmodium falciparums (Pf), Plasmodium vivax (Pv), ovum Shape plasmodium includes Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) two A subspecies, malariae (Pm), the DNA of Plasmodium knowlesi (Pk) carries out LAMP reaction, as a result as follows:
Plasmodium mtDNA gene
Toxoplasma, Schistosoma japonicum can not be expanded comprising the primer sets containing SEQ 1 to 6, to Plasmodium vivax, oval Plasmodium, malariae, Plasmodium knowlesi and plasmodium falciparum are effective.
Plasmodium falciparum ActinI gene
Comprising the primer sets containing SEQ 7 to 12 to toxoplasma, Schistosoma japonicum, Plasmodium vivax, Plasmodium ovale, three Day plasmodium, Plasmodium knowlesi can not expand, only effective to plasmodium falciparum.
Plasmodium vivax 18S rRNA gene
Comprising the primer sets containing SEQ 13 to 18 to toxoplasma, Schistosoma japonicum, Plasmodium ovale, malariae, Plasmodium knowlesi and plasmodium falciparum can not expand, only effective to Plasmodium vivax.
Plasmodium ovale SSU rRNA gene
Comprising the primer sets containing SEQ 19 to 24 to toxoplasma, Schistosoma japonicum, Plasmodium vivax, malariae, Plasmodium knowlesi and plasmodium falciparum can not expand, only effective to Plasmodium ovale.
Embodiment 4
Sensitivity tests
Protozoon counting is carried out to clinical sample, DNA is extracted and dilution, when parasitemia densities are 3.8 (2.0-7.0) It the use of the primer sets of SEQ 1 to 6 is the positive to the LAMP reaction result of five kinds of plasmodiums when Parasites/ μ L；Use SEQ 7 Primer sets to 12 are the positive to the LAMP reaction result of plasmodium falciparum；Using the primer sets of SEQ 13 to 18 to tertian fever original The LAMP reaction result of worm is the positive；The primer sets of SEQ 19 to 24 are the positive to the LAMP reaction result of Plasmodium ovale.
It constructs plasmid and converts Escherichia coli, DNA is extracted, by 109-101Doubling dilution, at the same carry out Nested-PCR with LAMP test, compares the detectable limit difference of two methods, analyzes its sensibility.As a result as follows:
Plasmodium mtDNA gene
It is tested using plasmid, the detectable limit of LAMP can achieve 102Copy/ μ L, and Nested-PCR can only reach To 104copy/μL。
Plasmodium falciparum ActinI gene
It is tested using plasmid, the detectable limit of LAMP can achieve 102Copy/ μ L, and Nested-PCR can only reach To 103copy/μL。
It is tested using malaria disease human blood sample, the detectable limit that Nested-PCR is reacted with LAMP is close, LAMP reaction It can reach 2.9 (95%CI 1.4-6.0) plasmodium/μ L blood.
Plasmodium vivax 18S rRNA gene
It is tested using plasmid, the detectable limit of LAMP can achieve 102Copy/ μ L, and Nested-PCR can only reach To 104copy/μL。
Plasmodium ovale SSU rRNA gene
It is tested using plasmid, the detectable limit of LAMP can achieve 103Copy/ μ L, and Nested-PCR can only reach To 104copy/μL。
The above results prove that the sensibility that our primer sets can monitor Infected With Plasmodium is higher than Nested-PCR.
Clinical sample
DNA extraction is carried out to malaria patient suspected's sample, while carrying out Nested-PCR and LAMP and testing.With clinical microscopy As a result laboratory standard Nested-PCR result generally acknowledged and at present compares, susceptibility, the specificity of analysis primer LAMP reaction Deng.
Patient's sample test result is as follows:
Plasmodium mtDNA gene
275 clinical malaria patient suspected's samples, nest-type PRC detect 237 malaria infections, the LAMP reaction of this group of primer Detection is also 237, as a result completely the same.
Plasmodium falciparum ActinI gene
466 clinical malaria patient suspected's sample nest-type PRCs detect 251 falciparum infections, this group of primer LAMP reaction 253 falciparum infections of detection, only 2 results are inconsistent.This 2 patients, control by clinical diagnostic It treats, makes a definite diagnosis malaria.
Plasmodium vivax 18S rRNA gene
273 clinical malaria patient suspected's sample nest-type PRCs detect 87 plasmodium vivax infections, the LAMP of this group of primer Reaction detection is also 87 plasmodium vivax infections, as a result completely the same.
Plasmodium ovale SSU rRNA gene
274 clinical malaria patient suspected's sample nest-type PRCs detect 58 Plasmodium ovale infection, the LAMP of this group of primer Reaction detection is 57 Plasmodium ovale infection, and as a result only 1 sample is inconsistent.
The primer for detecting/identifying Plasmodium and three kinds of plasmodiums using LAMP of the present inventor's research and development is more micro- Microscopy has higher sensitivity and higher specificity, and can more easily detect/reflect within a short period of time compared with PCR Determine the plasmodium in patient's blood sample.It include the primer sets containing SEQ 1 to 6 in embodiment 3, only to the effective of Plasmodium；Packet It is only effective to plasmodium falciparum containing the primer sets containing SEQ 7 to 12；Comprising the primer sets containing SEQ 13 to 18 only to every other day Plasmodium is effective；It is only to Plasmodium ovale effective comprising the primer sets containing SEQ 19 to 24, it was demonstrated that its specificity；Work as protozoon When density is lower, primer sets of the invention can also be detected, and be tested using plasmid, and the detectable limit of LAMP is higher than Nested-PCR。
In conclusion the primer sets according to the present invention for being used to detect Plasmodium and plasmodium kind, and use the primer Group separately or concurrently detects or identifies Plasmodium, plasmodium falciparum, Plasmodium vivax, malariae and Plasmodium ovale Method can carry out DNA amplification reaction by quick, easy and cheap water bath with thermostatic control especially in malaria endemic area so that A large amount of samples can be handled with cheap price.LAMP plasmodium detection primer group reaches the diagnosis requirement of clinical malaria, can drop Low its diagnoses cost, provides a new diagnostic method for clinical malaria.
SEQUENCE LISTING
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<120>plasmodium gene diagnostic primers
<130> 20190612
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<213>artificial synthesized
<400> 22
gcagcaggcg cgtaaattac aaccatgaaa tggccttgt 39
<210> 23
<211> 23
<212> DNA
<213>artificial synthesized
<400> 23
tctccggaat cgaactctaa ttc 23
<210> 24
<211> 25
<212> DNA
<213>artificial synthesized
<400> 24
tctaaagaag agaggtagtg acaag 25

Claims (6)

1. plasmodium gene diagnostic primers be used to or be respectively used to simultaneously detect or identify Plasmodium and/or plasmodium falciparum, The infection of one of Plasmodium vivax, Plasmodium ovale or a variety of plasmodiums；The primer is indicated containing SEQ 1 to 6 The oligonucleotides group of nucleic acid sequence, for expanding the specific region of Plasmodium mtDNA gene order；Contain 7 to 12 table of SEQ The oligonucleotides group for the nucleic acid sequence shown, the specific region for expanding plasmodium protozoon ActinI gene order；Contain SEQ The oligonucleotides group of 13 to 18 nucleic acid sequences indicated, for expanding the given zone of Plasmodium vivax 18S rRNA gene order Domain；Oligonucleotides group containing the nucleic acid sequence indicated of SEQ 19 to 24, for expanding Plasmodium ovale SSU rRNA gene sequence The specific region of column.
2. plasmodium gene diagnostic primers purposes according to claim 1 detects using primer sets or identifies Plasmodium Infection, the primer sets include the oligonucleotides group containing the nucleic acid sequence indicated of SEQ 1 to 6, for expanding Plasmodium The specific region of mtDNA gene order.
3. plasmodium gene diagnostic primers purposes according to claim 1 detects using primer sets or identifies malignant malaria original The infection of worm, which includes the oligonucleotides group containing the nucleic acid sequence indicated of SEQ 7 to 12, former for expanding plasmodium The specific region of worm ActinI gene order.
4. plasmodium gene diagnostic primers purposes according to claim 1 detects using primer sets or identifies tertian fever original The infection of worm, which includes the oligonucleotides group containing the nucleic acid sequence indicated of SEQ 13 to 18, for expanding tertian fever The specific region of protozoon 18S rRNA gene order.
5. plasmodium gene diagnostic primers purposes according to claim 1 detects using primer sets or identifies ovale malaria original The infection of worm, which includes the oligonucleotides group containing the nucleic acid sequence indicated of SEQ 19 to 24, for expanding ovale malaria The specific region of protozoon SSU rRNA gene order.
6. plasmodium gene diagnostic primers, it is characterised in that: the primer is the widow containing the nucleic acid sequence indicated of SEQ 1 to 6 Nucleotide group；Oligonucleotides group containing the nucleic acid sequence indicated of SEQ 7 to 12；The nucleic acid sequence indicated containing SEQ 13 to 18 Oligonucleotides group；Oligonucleotides group containing the nucleic acid sequence indicated of SEQ 19 to 24.
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